Mycobacterium tuberculosis Requires the ECF Sigma Factor SigE to Arrest Phagosome Maturation Stefano Casonato ¤ , Roberta Provvedi, Elisa Dainese, Giorgio Palu ` , Riccardo Manganelli* Department of Molecular Medicine, University of Padova, Padova, Italy Abstract SigE represents one of the best characterized alternative sigma factors of Mycobacterium tuberculosis, playing a major role in the response to several environmental stresses and essential for growth in macrophages and virulence. In previous work we demonstrated that a mutant of M. tuberculosis in which the sigE gene was disrupted by a cassette conferring hygromycin resistance is a promising vaccine candidate conferring better protection than Mycobacterium bovis BCG in a mouse model of infection. In this work we describe the construction of a new unmarked mutant in which the entire sigE gene was disrupted in order to fulfill the requirements of the Geneva consensus to enter clinical trials. After showing that the phenotype of this mutant is superimposable to that of the previous one, we further characterized the role of SigE in the M tuberculosis intracellular behavior showing that it is dispensable for replication in human pneumocytes, while it is essential for the arrest of phagosome maturation in THP-1-derived macrophages. Citation: Casonato S, Provvedi R, Dainese E, Palu ` G, Manganelli R (2014) Mycobacterium tuberculosis Requires the ECF Sigma Factor SigE to Arrest Phagosome Maturation. PLoS ONE 9(9): e108893. doi:10.1371/journal.pone.0108893 Editor: Joyoti Basu, Bose Institute, India Received June 12, 2014; Accepted September 4, 2014; Published September 30, 2014 Copyright: ß 2014 Casonato et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Data Availability: The authors confirm that all data underlying the findings are fully available without restriction. All relevant data are within the paper and its Supporting Information files. Funding: This work was supported by the European Community Seventh Framework Programme (FP7/2007–2013) under grant agreements 241745. The funder had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Competing Interests: Riccardo Manganelli is an Academic Editor of PLOS ONE. This does not alter our adherence to PLOS ONE Editorial policies and criteria. * Email: riccardo.manganelli@unipd.it ¤ Current address: Centre for Integrative Biology (CIBIO), University of Trento, Trento, Italy Introduction Tuberculosis (TB) represents a global public health problem and kills about 1.4 million people/year [1]. Current TB treatment requires a long and demanding multiple chemotherapy, while the only approved vaccine is the Bacille-Calmette-Guerin (BCG), an attenuated strain of Mycobacterium bovis that protects children from severe forms of TB, but not adults from pulmonary TB, failing to block the transmission chain [2]. The past decade has witnessed significant progress in the development of new TB vaccines, with the growth of a large and promising portfolio of candidates [3], but also the failure of the first phase II clinical trial of one of the most promising of them [4]. Moreover, the fundamental problem of the lack of reliable biomarkers to predict protection and disease progression still remained largely unsolved. In a previous work we have characterized an M. tuberculosis H37Rv mutant in which the gene encoding the alternative sigma factor SigE was disrupted by the insertion of a hygromycin- resistance cassette [5]. SigE is subject to a very complex regulation at transcriptional and posttranslational level [6] and is involved in the transcription of several genes following exposure to different stress conditions damaging the bacterial surface [5]. As a consequence, the sigE knock out mutant was more sensitive to various surface-disrupting stresses, such as the detergent sodium dodecyl sulfate (SDS) and the antibiotic vancomycin, and to various oxidative compounds. Moreover, it was unable to grow in resting THP-1-derived macrophages and human dendritic cells, was more sensitive to killing from activated mouse macrophages, and was severely attenuated in mice [7,8,9,10]. Due to its characteristics, we hypothesized that the sigE mutant could represent a promising live attenuated vaccine. Indeed, while its attenuation in mice was equal or better to that of BCG, it was more immunogenic and provided better protection from challenge with virulent M. tuberculosis [11]. In this work we describe the construction of a new H37Rv mutant in which the sigE gene was deleted without the introduction of a cassette conferring drug resistance, in order to fulfill the requirements to enter clinical trials [12]. After characterization of the new unmarked mutant, we provide proof that SigE is not required to growth in human pneumocytes, but is required to interfere with phagosome maturation. Materials and Methods Bacterial strains, growth media and transformation conditions The following bacterial strains were used: Escherichia coli Top10 (Invitrogen), E. coli DH5a and E. coli HB101 (laboratory stocks), M. tuberculosis H37Rv (laboratory stock). Escherichia coli strains were grown at 37uC in Luria-Bertani (LB) broth or on LB agar plates. Mycobacterial strains were grown at 37uC in Middlebrook 7H9 broth (BD) in 150 ml roller bottles with slow rotation (3 rpm) or 7H10 agar plates (BD), supplemented with 0.2% glycerol and 0.05% Tween-80. For growth of M. tuberculosis, the medium was supplemented with 10% ADN (Albumin, Dextrose, NaCl). When needed, antibiotics were added PLOS ONE | www.plosone.org 1 September 2014 | Volume 9 | Issue 9 | e108893